褪黑素腹腔注射对急性脑梗死大鼠神经细胞损伤凋亡的改善作用及其机制

Effects of intraperitoneal injection of melatonin on nerve cell injury and apoptosis in rats with acute cerebral infarction

目的观察褪黑素腹腔注射对急性脑梗死大鼠神经细胞损伤凋亡的改善作用,并探讨其机制。方法60只SD大鼠随机分为假手术组、模型组、褪黑素低剂量组、褪黑素中剂量组、褪黑素高剂量组、阳性对照组,每组10只。除假手术组外,其他5组建立急性脑梗死模型。造模成功后开始给药,褪黑素低剂量组、褪黑素中剂量组、褪黑素高剂量组分别给予褪黑素5、10、15 mg/kg,假手术组与模型组给予相同体积的生理盐水,阳性对照组给予尼莫地平15 mg/kg。给药方式均为腹腔注射,每日1次,共给药7 d。给药结束后24 h,采用Longa生物学评分法评估大鼠神经功能缺损情况,然后采用酶联免疫吸附法检测血清中氧化还原指标丙二醛(MDA)、超氧化物歧化酶(SOD)及炎性因子TNF-ɑ、IL-6、IL-1β。处死大鼠后分离脑组织,采用HE染色法观察各组脑组织病理学变化,采用TUNEL法检测大鼠脑组织神经细胞凋亡情况。采用Western Blotting法检测各组大鼠脑组织中Notch1/Hes1信号通路相关蛋白Notch1、Hes1。结果假手术组、模型组、褪黑素低剂量组、褪黑素中剂量组、褪黑素高剂量组、阳性对照组大鼠神经功能缺损评分分别为0、(3.91±0.63)、(3.23±0.52)、(2.67±0.45)、(1.84±0.47)、(1.66±0.43)分,其中褪黑素高剂量组大鼠神经功能缺损评分与阳性对照组相比,P>0.05,其余组间相比,P均<0.05。褪黑素高剂量组血清MDA、SOD、TNF-ɑ、IL-6、IL-1β水平与阳性对照组相比,P均>0.05;其余各组大鼠血清MDA、SOD、TNF-ɑ、IL-6、IL-1β水平组间相比,P均<0.05。与假手术组相比,模型组大鼠脑组织神经细胞受损严重,细胞分布稀疏凌乱,细胞空泡化严重,细胞核溶解现象严重;与模型组相比,褪黑素低剂量组、褪黑素中剂量组、褪黑素高剂量组、阳性对照组细胞病变现象减轻,褪黑素低剂量组与褪黑素中剂量组中少数细胞空泡化,细胞核溶解固缩现象减少,褪黑素高剂量组以及阳性对照组未见细胞核溶解固缩现象,细胞分布均匀整齐。假手术组、模型组、褪黑素低剂量组、褪黑素中剂量组、褪黑素高剂量组、阳性对照组大鼠脑组织神经细胞凋亡率分别为4.02%±0.67%、48.18%±5.23%、32.26%±3.24%、25.28%±2.63%、12.82%±1.56%、12.54%±1.23%,其中褪黑素高剂量组大鼠脑组织神经细胞凋亡率与阳性对照组相比,P>0.05,其余组间相比,P均<0.05。褪黑素高剂量组大鼠脑组织中Notch1、Hes1蛋白相对表达量与阳性对照组相比,P均>0.05;其余各组大鼠脑组织中Notch1、Hes1蛋白相对表达量组间相比,P均<0.05。结论高剂量(15 mg/kg)褪黑素腹腔注射可改善急性脑梗死大鼠的神经细胞损伤凋亡情况,褪黑素改善神经细胞损伤凋亡的机制可能与下调Notch1/Hes1信号通路相关蛋白Notch1、Hes1的表达有关。

Objective To explore the intervention effects of intraperitoneal injection of melatonin on nerve cell injury and apoptosis in rats with acute cerebral infarction,and to discuss their mechanism.Methods Sixty SD rats were randomly grouped into the sham operation group,model group,low-dose,medium-dose,high-dose melatonin groups,and positive control group,with 10 in each group.Except for the sham operation group,we established rat models of acute cerebral infarction in the other five groups.After successful modeling,we started the drug administration,and the rats in the low-dose melatonin group,the medium-dose melatonin group,and the high-dose melatonin group were given 5,10,and15 mg/kg melatonin,respectively,the rats in the sham operation group and the model group were given the same volume of normal saline,and the rats in the positive control group were given nimodipine at a dose of 15 mg/kg.The methods of administration were intraperitoneal injection,once a day,for a total of 7 days.At 24 h after the end of administration,the neurological deficits of rats were evaluated according to the Longa biological scoring method,then the redox indexes malondialdehyde(MDA),superoxide dismutase(SOD) and inflammatory factors TNF-α,IL-6 and IL-1β in serum were measured by enzyme-linked immunosorbent assay.After the rats were sacrificed,HE staining method was performed to observe the pathological changes of the brain tissues in each group,and the TUNEL method was performed to detect the neuronal apoptosis in the rat brain tissues.Western blotting was performed to measure Notch1/Hes1 signaling pathway-related proteins Notch1 and Hes1 in the brain tissues of rats in each group.Results The neurological deficit scores of rats in the sham operation group,model group,low-dose melatonin group,medium-dose melatonin group,high-dose melatonin group,and positive control group were 0,(3.91±0.63),(3.23±0.52),(2.67±0.45),(1.84±0.47),and(1.66±0.43) points,respectively;no significant difference was found in the neurological deficit score between the high-dose melatonin group and positive control group(P>0.05),but significant difference was found among other groups(all P<0.05).No significant difference was found in the levels of serum MDA,SOD,TNF-α,IL-6 or IL-1β between the high-dose melatonin group and the positive control group(all P>0.05),but there was significant difference in the levels of serum MDA,SOD,TNF-α,IL-6 and IL-1β among the other groups(all P<0.05).Compared with the sham operation group,the nerve cells in the brain tissues of rats in the model group were severely damaged,the distribution of cells was sparse and disordered,the cells showed vacuolation,and the nuclei were severely dissolved.Compared with the model group,the lowdose melatonin group,the medium-dose melatonin group,the high-dose melatonin group,and the positive control group had less cytopathic lesions;in the low-dose melatonin group and the medium-dose melatonin group,a small number of cells showed vacuolation,and the pyknosis of nuclei was reduced;in the high-dose melatonin group and the positive control group,no nuclear pyknosis was observed,and the cells were evenly distributed.The neuronal apoptosis rates in the sham operation group,model group,low-dose melatonin group,medium-dose melatonin group,high-dose melatonin group,and positive control group were 4.02%±0.67%,48.18%±5.23%,32.26%±3.24%,25.28%±2.63%,12.82%±1.56%,and 12.54%±1.23%,respectively,with no statistically significant difference between the high-dose melatonin group and the positive control group(P>0.05),but there was significant difference among the other groups(all P<0.05).There were no statistically significant differences in the relative expression levels of Notch1 and Hes1 proteins in the brain tissue of rats between the high-dose melatonin group and the positive control group(both P>0.05);and significant difference was found in the relative expression levels of Notch1 and Hes1 proteins in the brain tissues of rats among the other groups(all P<0.05).Conclusions Intraperitoneal injection of high-dose(15 mg/kg) melatonin can alleviate the injury and apoptosis of nerve cells in rats with acute cerebral infarction.The mechanism may be related to the down-regulated expression of Notch1/Hes1 signaling pathway-related Notch1 and Hes1 proteins.